Secretion of granules from cultured RBL-2H3 mast cells are dependent on signals generated through a rise in cytosolic calcium and activation of protein kinase C (PKC). Phosphorylation of myosin heavy and light chains by PKC and Ca2+-dependent myosin light chain kinase are temporally correlated with degranulation of RBL-2H3 cells to indicate possible regulation of degranulation by myosin. Additional potential regulators of degranulation that we have characterized in RBL-2H3 cells include proteins of the SNARE complex (see last years report) and the Gi family of GTP-binding proteins which are activated directly by polybasic mast cell secretagogues (Science 262:1969, 1993). This year we have focussed on the latter proteins and found that cultured RBL-2H3 mast cells, which do not normally respond to these secretagogues, do so after prolonged exposure to the kinase inhibitor, quercetin. This inhibitor induces phenotypic changes in these cells accompanied by a substantial increase (> 7-fold) in expression of alpha-subunits of the pertussis toxin-sensitive G proteins, Gi-2 and Gi-3. As exemplified by studies with one of these secretagogues, compound 48/80, secretion is associated with transient hydrolysis of phosphoinositides and a transient increase in cytosolic calcium ions. These responses are inhibited by pertussis toxin and, in addition, secretion is blocked by calcium chelation and the protein kinase C inhibitor, Ro31-7549. These results delineate a pathway for compound 48/80-induced secretion in mast cells via Gi protein(s), phospholipase C, calcium and protein kinase C. The results also imply that phospholipase C, most likely phospholipase Cbeta3, can be transiently activated by subunits of Gi proteins to induce cellular responses. Of historic interest, compound 48/80 (so named because it was the 80th compound of a series synthesized in 1948 by Burroughs Welcome), has been actively investigated since the discovery 50 years ago that it caused mast cell degranulation instead of the intended smooth muscle relaxation. Its actions lead to the important discoveries that mast cells were the predominant source of histamine and were responsible for anaphylactic reactions. Recent interest has focussed on its activation of Gi proteins but the connections to secretion were unclear. Our unexpected finding that quercetin markedly increases expression of Gi-2 and Gi-3 and sensitizes RBL-2H3 cells to compound 48/80, has allowed us to define for the first time the signaling pathway for 48/80-induced secretion.